This invention relates to the field of antimicrobial agents and more specifically it relates to the use of milbemycins and analogous compounds as efflux pump inhibitors to be co-administered with antimicrobial agents to inhibit the growth of microbial cells.
Fungal infections are relatively rare in immunocompetent humans or other mammals. A number of Candida species are often present as benign commensal organisms in the digestive system of healthy individuals (Shepherd, M et al, Ann. Rev. Microbiol., 39:579-614, 1985). Fungal infections, however, can be life threatening for immunocompromised individuals. Three major groups of severely immunocompromised individuals have emerged in recent years, These are: 1) cancer patients undergoing chemotherapy, 2) organ transplant patients treated with immunosuppressive agents, and 3)AIDS patients. The frequency of fungal infections has risen world-wide in recent years. Data from the National Nosocomial Infections Surveillance System conducted in the United States showed 487 percent increase in Candida bloodstream infections between 1980 and 1989 (Reviewed in Rex, M. Rinldi, and M. Pfaller. 1995. Resistance of Candida species to fluconazole. Antimicrob. Agents and Chemother. 39:1-8). Oropharyngeal candidiasis was shown to be the most common fungal infection in AIDS patients. Prevalence studies have suggested that up to 90% of patients with AIDS have had at least one episode of oropharyngeal candidiasis (Powderly. 1994. Resistant Candidiasis. 1994. AIDS research and Human Retrovirusses. 10:925-929).
There are only a small number of therapeutic agents available for the treatment of serious fungal infections. Amphotericin B, flucytosine, fluconazole, itraconazole and ketoconazole are some of the few currently available drugs against systemic fungal infections (Odds. 1993. Resistance of yeasts to azole-derivative antifungals. J. Antimicrob. Chemother. 31: 463-471).
The mechanism of action of amphotericin B, a polyene macrolide antibiotic, is based on its interaction with the plasma membrane of sensitive organisms, which impairs the barrier function of the membrane. Its selectivity may be related to its greater affinity for the ergosterol of fungal membranes than for the cholesterol of mammalian membranes. Amphotericin B is a fungicidal agent. Resistance to amphotericin B is rare and is based on a marked (74-85%) decrease in the ergosterol content in resistant variants. However, amphotericin B is associated with many toxic side effects and is poorly absorbed from the gastrointestinal tract, which necessitates intravenous administration.
The mechanism of action of flucytosine (5FC) is the inhibition of DNA and RNA synthesis. 5FC is taken up by a cytosine permease. It is converted to 5-fluorouracil (5FU) by cytosine deaminase inside the fungal cell. The low activity of cytosine deaminase in mammalian cells is the basis for the low toxicity of 5FC in human. 5FU is converted into 5-fluorouridilic acid, which is further phosphorylated and incorporated into RNA. As a result of formation of such aberrant RNA the fungal growth is inhibited. 5FU is also converted to a potent inhibitor of thymidilate synthase and, as a result, inhibits DNA synthesis. Thus, 5FC is a potent fungicidal agent. However, it rapidly becomes ineffective since mutations to resistance arise with high frequency. This resistance results from loss or mutation of any of the enzymes which are involved in its conversion into toxic intermediates for RNA and DNA synthesis (Reviewed in Vanden Bossche, P. Marickal, and F. Odds. 1994. Molecular mechanisms of drug resistance in fungi. Trends Microbiol. 2:393-400).
Azoles (e.g., fluconazole, itraconazole and ketoconazole) are currently the most important agents for the treatment of fungal diseases. The primary mechanism of action of azole antifungals is inhibition of ergosterol biosynthesis. In azole-treated cells there is accumulation of 14xcex1-methyl-sterols, the precursor intermediates of ergosterol. Conversion of 14xcex1-methyl-sterols to ergosterol was shown to be dependent on cytochrome P-450. Azoles bind to P-450 and inhibit the function of this enzyme. (Reviewed in Saag and W. Dismukes. 1988. Azole antifungal agents: emphesis on new triazoles. Antimicrob. Agents Chemother. 32:1-8). The first oral azole which was proven to be effective in mycoses was clotrimazole. However, brief treatment with clotrimazole rapidly induces liver microsomal enzymes which increases metabolism of the drug and diminishes its antifungal activity. Another azole, miconazole, is not rapidly metabolized, but it has multiple toxic effects. As a result, it has very limited use as a topical agent for cutaneous mycoses. Ketoconazole was developed in the late 1970s. It was the first azole that could be given orally for systemic use and for some time it was the most important azole antifungal agent. It is less toxic than miconazole, however, dose-related inhibition of testosterone synthesis may result in menstrual irregularities, sexual impotence or oligospermia (Saag and W. Dismukes. 1988. Azole antifungal agents: emphasis on new triazoles. Antimicrob. Agents Chemother. 32:1-8). Relatively recently, two new azole, fluconazole and itraconazole, have been developed.
Fluconazole is currently the most extensively used agent for the treatment of patients with severe candidiasis. It has several advantages over the earlier azole antifungals, including ketoconazole. It has higher solubility in water, longer plasma-half-life, and relatively low toxicity. The bioavailability of fluconazole after oral administration is 90%. Between 1988 and 1993, fluconazole was used to treat over 15 million patients, including at least 250,000 AIDS patients (Hitchcock, C. A. 1993. Resistance of Candida albicans to azole antifungal agents. Biochem Soc. Trans. 21:1039-1047), and fluconazole treatment of patients with oropharyngeal candidiasis has been adopted by many clinics. (Recently licensed itraconazole is not used as extensively because of its much lower bioavailability).
Due to wide use of fluconazole for both treatment (and in many cases, this treatment is continued over long periods of time) and prophylaxis, reports of failure of therapy due to appearance of Candida which are resistant to fluconazole began to appear (reviewed in Rex, M. Rinldi, and M. Pfaller. 1995. Resistance of Candida species to fluconazole. Antimicrob. Agents and Chemother. 39:1-8, Vanden Bossche, P. Marickal, and F. Odds. 1994. Molecular mechanisms of drug resistance in fungi. Trends Microbiol. 2:393-400). Three different routes of acquisition of resistant variants were described. In the first scenario, infecting Candida albicans (C. albicans) were initially susceptible, but mutated and become resistant. Mutants resistant to fluconazole only and mutants which are cross-resistant to other azoles (ketoconazole, itraconazole) have been isolated. In the second scenario, patients were initially colonized with fluconazole resistant C. albicans. In the third scenario, the recent widespread use of fluconazole led to a rise in the prevalence of colonization and infection by other Candida species, such as Candida glabrata and Candida krusei, which are intrinsically less susceptible to fluconazole (reviewed in Odds. 1993. Resistans of yeasts to azole-derivative antifungals. J. Antimicrob. Chemother. 31: 463-471).
A limited number of studies on the mechanism of resistance to fluconazole in clinical isolates have appeared in the literature. It was shown that in one, probably exceptional case, amplification of the gene CYP51, encoding P-450 (fluconazole target) is implicated in drug resistance (Vanden Bossche, P. Marickal, F. Odds, L. Le Jeune, and M.-C. Coene. 1992. Characterization of an azole resistant Candida albicans isolate. Antimicrob. Agents and Chemother. 36: 2602-2610). In another case resistance to fluconazole was correlated with the appearance of an altered P-450 which had decreased affinity to fluconazole (Hitchcock, C. A. 1993. Resistance of Candida albicans to azole antifungal agents. Biochem Soc. Trans. 21:1039-1047). However, the majority of the cases of fluconazole resistance appears to originate from decreased accumulation of fluconazole inside the resistant cells (H. Vanden Bossche et al, 1994, F. C. Odds, 1993). Moreover, decreased accumulation of fluconazole in resistant cells was also demonstrated for the other two types of resistance described above. It was also reported that species which are intrinsically non-susceptible to fluconazole, such as C. glabrata and C. krusei, and Aspergillus fumigatus accumulate less fluconazole than C. albicans (H. Vanden Bossche et al, 1994). C. glabrata and C. krusei are much more susceptible to itraconazole, and it has been shown that these two fungal species accumulate higher levels of itraconazole than fluconazole (Marichal et al., 1995, Origin of differences in susceptibility of Candida krusei to azole antifungal agents, Mycoses 38:111-117). Thus, it appears that both the acquired resistance and the intrinsic non-susceptibility of different fungal species were due to the decreased drug accumulation in the cell. The reason for decreased drug accumulation can be either decreased uptake and/or increased efflux from the cell.
Active efflux is associated with activity of membrane transporter proteins, also known as efflux pumps. These pumps are ubiquitous from bacteria to mammals (for review, see Higgins, C. F. 1992. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8:67-113). For their activity they can utilize either the energy of ATP hydrolysis (ABC-transporters superfamily) or the energy of the proton transfer (Major Facilitators, MF, superfamily). They can be specific toward a particular substrate as in the case of the TetA protein from gram-negative bacteria which effluxes tetracycline, the MsrA protein from Staphylococcus aureus (S. aureus) which effluxes erythromycin and related macrolides and the efflux pumps which extrude antibiotics from antibiotic-producing organisms.
On the other hand, there are a large number of efflux pumps which are capable of extruding from the cell a variety of structurally unrelated compounds. Many of these pumps are of considerable clinical significance: mammalian P-glycoprotein multi-drug resistant efflux pump confers resistance to cancer cells against chemotherapeutic drugs (reviewed in Gottesman, M and Ira Pastan. 1993. Biochemistry of multi-drug resistance mediated by the multidrug transporter. Annu. Rev. Biochem 62:385-427), Pgh1 has a role in the chloroquine resistance of the malaria parasite (Borst and M. Quelette. 1995. New mechanisms of drug resistance in parasitic protozoa. Annu. Rev. Microbiol. 49:427-60), Mex pumps confer resistance to Pseudomonas aeruginosa (P. aeruginosa) against quinolones and many others structurally unrelated antibiotics (reviewed in Nikaido, 1994, Prevention of drug access to bacterial targets: permeability barriers and active efflux, Science 264:382-388). Recently, multiple-drug resistant (MDR) pumps were implicated in fluconazole resistance in both C. albicans and C. glabrata (Parkinson et al. 1995. Fluconazole resistance due to energy-dependent drug efflux in Candida glabrata. Antimicrob. Agents Chemother. 39:1696-1699; Sanglard et al. 1995. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob. Agents Chemother. 39:2378-2386; Albertson et al. 1996. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob. Agents Chemother. 40:2835-2841).
The information provided and the references cited herein are not admitted to be prior art to the present invention, but are provided solely to assist the understanding of the reader.
This invention is concerned with the use of the milbemycins and related compounds as efflux pump inhibitors and methods for treating microbial infections or cancer or other condition using the milbemycins and related compounds. Particularly appropriate examples of such microbial infections are infections caused by pathogenic fungal species, Candida albicans, Candida glabrata, and Candida krusei which are resistant to many of the commonly used antifungal agents, such as a variety of azoles. While C. albicans, C. glabrata, and C. krusei are examples of appropriate fungi, other fungal and microbial species may contain similar broad substrate efflux pumps, which actively export a variety of antimicrobial agents, and thus are also appropriate targets. The invention further concerns the identification and use of efflux pump inhibitors targeted to a CDR1, CDR2, BEN, or FLU1 efflux pump or a homolog of such a pump.
The term xe2x80x9cefflux pumpxe2x80x9d refers to a protein assembly which exports substrate molecules from the cytoplasm or periplasm of a cell, in an energy dependent fashion. An xe2x80x9cefflux pump inhibitorxe2x80x9d is a compound which specifically interferes with the ability of an efflux pump to export its normal substrate or other compounds such as antimicrobial agents. Efflux pump inhibitors are useful, for example, for treating microbial infections by reducing the export of a co-administered antimicrobial agent. Thus, a microbe which can efflux an antimicrobial agent is resistant to the inhibiting effect of the antimicrobial agent, and becomes sensitive to the inhibiting effect of the same antimicrobial agent in the presence of an efflux pump inhibitor. An efflux pump inhibitor is thus distinguished from generally toxic compounds, general metabolic poisons, energy uncouplers, or other such compounds which have many direct inhibitory effects in a cell. It is recognized that an efflux pump inhibitor may have additional indirect effects as inhibition of efflux pump activity alters the intracellular levels of certain species of compounds or ions.
In connection with efflux pumps, the term xe2x80x9chomologxe2x80x9d refers to another efflux pump which has significant amino acid sequence homology as determined by optimized alignment comparison of the polypeptide sequence or sequences from the homolog with the reference efflux pump polypeptide sequence or sequences. Those skilled in the art are familiar with such methods and with the computer programs generally used for performing such comparisons. Preferably the homolog has at least 20% amino acid sequence identity, more preferably at least 30%, or preferably at least 30%, or at least 40% sequence similarity, and more preferably at least 50%, as determined using any widely accepted sequence analysis method as known to those skilled in the art.
The term xe2x80x9cmilbemycinxe2x80x9d or xe2x80x9cmilbemycinsxe2x80x9d refers to a group of sixteen-membered macrolactones containing a fused spiro ether moiety, for example, compounds of the type isolated from the fermentation of Streptomyces hygroscopicus NRRL 5739, as referenced below. Milbemycins have been described as having insecticidal, acaracidal, and anthemlmintic activity. A variety of other milbemycins and milbemycin-type compounds are described herein and in references listed below. Yet others are known to those skilled in the art.
The term xe2x80x9cmilbemycin-type compoundxe2x80x9d refers to compounds and families of compounds structurally related to milbemycins which are also sixteen-membered macrolactones containing a fused spiro ether moiety and which also have efflux pump inhibitory activity. Such compounds also generally have insecticidal, acaracidal, or anthelmintic actitities. Such compounds have been isolated from the fermentation of microorganisms, and include compounds modified from such natural products by chemical modification. Thus, these compounds include milbemycins, compounds structurally related to milbemycins, and derivatives of such compounds. Preferred milbemycin-type compounds are not avermectins.
In connection with the milbemycin-type compounds, the term xe2x80x9cderivativexe2x80x9d refers to a compound which has been chemically modified from another milbelycin or milbemycin-type compound by the addition, substitution, or modification of a substituent of the starting compound, and which retains or has gained efflux pump inhibitory activity. Examples of preferred substituents are given in certain of the references cited herein in connection with milbemycin-type compounds.
In a first aspect, the invention provides a method for inhibiting the growth of cells, preferably inhibiting growth of a microbe, preferably a fungus, by contacting the microbe with a milbemycin or milbemycin-type compound or derivative, preferably with a milbemycin or derivative, and a second compound. The milbemycin or milbemycin-type compound enhances the susceptibility of the cells, e.g., the microbe, to the second compound. Preferably the second compound is an antimicrobial agent, such as an antifungal agent, for example, an azole such as fluconazole, or terbinafine. Preferably the milbemycin or milbemycin-type compound or derivative is active on at least one of a CDR1, CDR2, BEN, or FLU1 efflux pump or a homolog of such a pump. In preferred embodiments, the compound is active on a plurality of those efflux pumps.
In preferred embodiments, the cells can be contacted with the milbemycin or milbemycin-type compound or derivative and the second compound simultaneously or can be contacted serially.
The cells can be of a variety of different types, so long as the milbemycin or milbemycin-type compound enhances the susceptibility of the cells to the second compound. For example, the cells can be animal cells, e.g., human cells, lower eukaryotic cells, or prokaryotic cells. In preferred embodiments, the cells are of a microbe, preferably a fungus, such as a Candida species, for example, Candida albicans, Candida krusei, or Candida glabrata. In preferred embodiments, the cells are mammalian cells, e.g., human cells, which preferably express P-glycoprotein. The cells may be cancer cells, in which case the second compound is preferably an anticancer agent.
The term xe2x80x9cmicrobexe2x80x9d is used in its usual biological sense to refer to very small organisms, which generally are only readily observable when viewed under a microscope or when aggregated. Thus, the organism is generally of less than 1 mm in average dimension, more typically less than 100 xcexcm, and often less than 10 xcexcm. However, it is understood that certain microbes have such size only during certain stages of the life cycle. The term xe2x80x9cmicrobexe2x80x9d is also meant to include fungi which have a mycelial vegetative stage. In this case, the term xe2x80x9cmicrobial cellxe2x80x9d can refer to a coenocytic or mycelial structure, which is generally polynucleate. Thus, the term xe2x80x9cmicrobexe2x80x9d includes, for example, bacteria, algae, fungi, and protozoans.
The terms xe2x80x9cfungusxe2x80x9d and xe2x80x9cfungixe2x80x9d refer to lower eukaryotic organisms as generally understood by those skilled in the art. Commonly fungi have a mycelial or coenocytic vegetative stage. However, in the context of this invention, unless specifically indicated to the contrary, included are the yeasts (e.g., Saccharomyces species). In this context, xe2x80x9cyeastxe2x80x9d refers to a lower eukaryotic organism which has a single celled growth stage and is classified within the fungi, for example, based on properties such as cell structure, reproductive mechanisms, nucleic acid sequence comparisons or other characteristics commonly utilized for classifying organisms. The fungi include the following classes: Zygomycetes, Ascomycetes, the Basidiomycetes, Deuteromycetes, and Oomycetes.
The invention encompasses, in the various aspects, the use of a variety of milbemycins and milbemycin-type compounds. In particular embodiments, these include compounds described by the structures provided below and compounds described in the references indicated below in connection with compounds appropriate for this invention. These include, for example, the S-541 compounds, the LL-F28249 compounds, the N787-182 compounds, and the VM series compounds. These groups of compounds are identified in the references cited below and in the attached figures. In addition, further chemical derivatives of these compounds which retain efflux inhibiting activity are within the scope of this invention.
The second compound is generally selected to be one which has a phamacologic activity in the cell. Thus, in embodiments where the cell is a mammalian cell, e.g., a human cell, the second compound can be an anticancer agent. Clearly, this is most appropriate where the mammalian cells are cancer cells. Likewise, where the cells are microbial cells, e.g., fungal cells, the second compound can be an antimicrobial agent, e.g., an antifungal agent.
In general, the method involves the inhibition of an efflux pump by the milbemycin or milbemycin-type compound in a case where the efflux pump would otherwise export the second compound. Thus, in preferred embodiments, the method is used in connection with a microbe or other cell which is resistant to the second compound, as the milbemycin or milbemycin-type compound will enhance the susceptibility of the cell for that second compound.
In this context, the term xe2x80x9cresistantxe2x80x9d refers to cells, e.g., fungi, which are not susceptible to the effects of a particular compound such as an antifungal or anticancer agent. Resistance can be intrinsic or acquired. Intrinsic resistance is the naturally occurring resistance that is found among most or all members of an entire species of organism, e.g., microorganism. Acquired resistance occurs through mutations of existing DNA or acquisition of new DNA in a previously susceptible organism. For example, antimicrobial agents may exert selective pressure for selection of acquired resistance. Both intrinsic and acquired resistance may be mediated by a variety of different mechanisms. A mechanism of particular interest in the present invention is resistance mediated by efficient export of potentially therapeutic agents, e.g., antimicrobial agents, from the cell, e.g., microbial cell. Thus, a resistant cell can be identified as one which is less susceptible to a particular therapeutic compound (e.g., antimicrobial agent or anticancer agent) than is a cell which does not contain a particular cellular mechanism for reducing the susceptibility of the cell to that compound. Thus, for example, for resistance mediated by expression of one or more efflux pumps which export a particular compound, the resistant cell can be identified by comparison of the susceptibility of the cell for the compound as compared to an otherwise isogeneic cell which does not express a functional efflux pump which exports the compound. Preferably, but not necessarily, a resistant cell is at least 2-fold, more preferably at least 4-fold, and most preferably at least 8-fold less susceptible to effects of a compound than are cells which are isogeneic except for the absence of the corresponding functional resistance mechanism or mechanisms.
The milbemycin or milbemycin-type compound can be prepared in various ways. These include the isolation of a natural product, such as isolation from a fermentation of one of the strains identified in the references listed below. As understood by those skilled in the art, certain of the compounds may be prepared by directed biosynthesis. In addition, the milbemycin or milbemycin-type compound can be prepared by chemical modification or microbial transformation of a naturally-occurring milbemycin or milbemycin-type compound. Likewise, the milbemycin or milbemycin-type compound can be prepared by chemical synthesis.
xe2x80x9cDirected biosynthesisxe2x80x9d refers to a method for causing biosynthesis of a product or products by the addition of an appropriate biosynthetic precursor or inhibitor of a particular biosynthetic pathway to a microbial fermentation. This causes the microorganism to incorporate the added precursor into a secondary metabolite (e.g., a milbemycin) it normally produces or to change the chemical structure of the secondary metabolite by omitting some biosynthetic step which would normally be performed. Milbemycin-type compounds which result from such directed biosynthesis are within the scope of this invention.
xe2x80x9cMicrobial transformationxe2x80x9d refers to the modification of the structure of a chemical compound (e.g., a milbemycin-type compound) by the fermentation process of a microorganism. Such transformations are known in the art, and are described, for example, in Chen, EP 369,502, EP 475,518, and EP 478,064. Milbemycin-type compounds which result from such transformation are within the scope of this invention.
In a related aspect or in embodiments of the above aspect, the invention provides a method for inhibiting the growth of a mammalian cell by contacting the cell with a milbemycin or milbemycin-type compound and a second compound. Similar to the microbial cells as described above, the milbemycin or milbemycin-type compound enhances the susceptibility of the cell to the second compound. This method is particularly applicable to inhibition of growth of a cancer cell, preferably where the second compound is an anticancer agent. This is particularly useful when the cancer cell is resistant to the anticancer agent. In particular it was shown that the milbemycin-type compounds inhibit the activity of P-glycoprotein, and thus the method is preferably used to inhibit the growth of cells which express or overexpress P-glycoprotein.
Also, as indicated above, the methods of inhibiting cells, methods of treatment, compositions, and methods of preparing compositions of this invention can use a variety of milbemycins or milbemycin-type compounds, such as those described below.
Such appropriate compounds include milbemycin-type macrolides of Structures (I), (II), and (III) 
Where independently
for Structures I and II R1=O, OH, OCH3;
for Structure III R1=OH;
R2=CH3, CH2OCOCH2CH2CH3, CH2OCOCH(CH3)2, CH2OCOCH2CH2CH2CH3, CH2OCOCH2CH(CH3)2, CH2OCOCH2CH(CH3)CH2CH3, CH2OCOCH2CH(CH2CH3)2, CH2OCOCHxe2x95x90CHCH3, CH2OCOCHxe2x95x90C(CH3)2, CH2OCOCHxe2x95x90C(CH3)(CH2CH3), CH2OCOCHxe2x95x90C(CH2CH3)2, CH2OCOC(CH3)xe2x95x90CHCH3, CH2O2CCH2C6H5, or 
R3=CH3, CH2CH3, CH(CH3)2, CH2C(CH3)CH2CH3, C(CH3)xe2x95x90CH(CH3), C(CH3)xe2x95x90CH(CH2CH3), C(CH3)xe2x95x90CH(CH(CH3)2), C(CH3)xe2x95x90CH(CH3);
R4=CH3, CH2CH3;
R5=H, OH, OCOCH(CH3)2, OCOCH(CH3)Bun, OCOC4H9; OCOC6H13;
R6=H, OH;
R7=H, OH, OCOCH(CH3)2, OCOCH2CH(CH3)2;
for Structure II
R8=CH3, CH2OH, CHO;
R9=H, OH.
Further, the following macrolide antibiotics, milbemycins (also referenced as B-41 compounds) (J. Antibiotics, 33:1120-1127, 1980; J. Antibiotics, 36:980-990, 1983; Acki et al, U.S. Pat. No. 3,950,360, 1976, U.S. Pat. No. 3,992,551, 1976), the S-541 compounds (Tetrahedron Lett, 28:5353-5356, 1987; Eur. Pat. Appl. 242,052, 1987; Ward et al., Brit. Pat. 2,166,436, 1986), the LL-F28249 compounds (J. Antibiotics, 41:519-529, 1988; Carter et al, U.S. Pat. No. 5,106,994, 1992; U.S. Pat. No. 5,169,956, 1992), the N 787-182 compounds (J. Antibiotics, 45:659-670, 1992; Haxell et al., Eur. Pat. Appl. 334,484, 1989;), and the VM series compounds (J. Antibiotics, 43:1069-1076, 1990; J. Antibiotics, 49:272-280, 1996; Banks et al, Eur. Pat. Appl. 254,583, 1987; Banks et al., Eur. Pat. Appl. 325,462, 1989) are efflux pump inhibitors and can be utilized in embodiments of the various aspects of this invention.
Additional milbemycins and milbemycin-type compounds and derivatives appropriate for use in this invention are described in the following patents and publications:
DE 3916931 A1, 1990, Sollner et al.
GB 2,170,499 B, 1985, Poole et al.
EP 242,052 A, 1987, Rudd et al.
GB 2,187,742, 1987, Ramsay et al.
AU 23,602, 1988
EP 298,423 A, 1988, Katoh et al.
EP 204,421 A, 1986, Goegelman
EP 205,251 A, 1986, Goegelman
EP 300,674 A, 1989, Chen
EP 369,502 A, 1989, Chen
EP 475,518 A, 1991, Arison et al.
EP 511,881 A, 1992, Goegelman et al.
EP 478,064 A, 1991, Shumanov and White
EP 308,145 A, 1989, Dutton and Perry
EP 410,615 A, 1990, Hiroshi et al.
JP 63-264484, 1988
JP 63-227590, 1988
Tetrahedron Lett, 28:5353-5356, 1987, Ramsay et al.
J. Antibiotics, 43:1321-1329, 1990, Nakagawa et al.
U.S. Pat. No. 5,030,650
U.S. Pat. No. 4,916,154
U.S. Pat. No. 4,886,829
EP 280,928
U.S. Pat. No. 5,149,832
U.S. Pat. No. 4,988,824
GB 2,167,751
U.S. Pat. No. 4,547,520
AU 8,317,168
EP 444,964
U.S. Pat. No. 4,093,629
U.S. Pat. No. 4,134,973
U.S. Pat. No. 4,144,352
The structures of some of the compounds described in the above references are shown in the attached figures. The references cited above dealing with milbemycins and milbemycin-type compounds and derivatives are further indicative of the level of skill in the art concerning obtaining and modifying those compounds. In particular, the references provide source organisms, fermentation methods and conditions, and purification methods, as well as modification processes, as known to those skilled in the art.
Compounds prepared by semi-synthetic modifications, enzymetic modifications and microbial transformations of the compounds described in the above patents and publications are also appropriate for use in this invention. Compounds prepared by directed biosynthesis using the producing strains which produce the compounds described in the above patents and publications are also within the scope of this invention.
In the context of cell growth, the term xe2x80x9cinhibitxe2x80x9d means that the rate of growth of the cell, e.g., the microbial population, is decreased. Such inhibition can be monitored, for example, by the difference in turbidity of liquid cultures in the presence or absence of the inhibiting agent, or by the difference in plaque size for cultures on solid media in the presence or absence of the inhibiting agent, or by other methods well-known to those skilled in the art.
In reference to the presence of a specific efflux pump in a fungus (similarly for other microbes or cells), the term xe2x80x9coverproducesxe2x80x9d refers to the presence in that fungus of a significantly larger number of the specific efflux pump than is found in most naturally occurring (usually non-hospital varieties) isolates of that fungal species. The term does not refer merely to the production of a large number of the component polypeptides of an efflux pump, but rather to the presence of a larger number of functional efflux pumps in the membranes of the cell. Consequently, a fungal cell which overproduces an efflux pump, will export the substrate molecules more efficiently than a strain of that fungus which does not overproduce the efflux pump.
A fungal strain which overproduces an efflux pump, is thus in contrast to a xe2x80x9cwild-type strainxe2x80x9d. A wild-type strain produces a specific efflux pump at a level which is typical of natural isolates of that fungal species. More importantly, however, a wild-type strain produces a specific efflux pump at a level which is significantly lower than a related strain which overproduces that specific efflux pump.
As used herein, the term xe2x80x9cantifungal agentxe2x80x9d refers to a compound which specifically inhibits the growth of a fungus. More generally, the term xe2x80x9cantimicrobial agentxe2x80x9d refers to a compound which specifically inhibits the growth of a microbe, thus the explanation of this term applies also to other microbes and antimicrobial agents. Thus such agents may have either fungicidal or fungistatic activity. In general, if an antifungal agent is fungistatic, it means that the agent essentially stops fungal cell growth (but does not kill the fungus); if the agent is fungicidal, it means that the agent kills the fungal cells (and may stop growth before killing the fungus). However the term specifically distinguishes compounds which are generally toxic to cells. Some examples of different classes of antifungal agents are amphotericin B, flucytosine, azoles such as fluconazole, itraconazole, and ketoconazole, and terbinafine. The efflux pump inhibitors of this invention may be antimicrobial (e.g., antifungal) agents when used alone, and/or they may potentiate the activity of another antimicrobial agent (increase the susceptibility of the microbe for that other antimicrobial agent).
A xe2x80x9csub-inhibitory concentrationxe2x80x9d of an inhibitor, e.g., an antifungal agent, is a concentration which is greater than zero, but less than the concentration which would inhibit the majority of the cells in a population, e.g., a fungal population, of that specific fungal strain. (Similarly for other microbes.) In general, a sub-inhibitory concentration of an antifungal agent (or antimicrobial agent) is a concentration less than the Minimum Inhibitory Concentration (MIC).
In a related aspect, this invention provides a method for treating a microbial infection, preferably a fungal infection, in an organism, such as in an animal or a plant. Animals specifically include mammals, e.g., humans. The methods involves treating an animal or other organism bearing or suffering from such an infection with an antimicrobial agent, preferably an antifungal agent, and an efflux pump inhibitor which increase the susceptibility of the microbe, e.g., fungus, for that agent (e.g., antifungal agent). In this way, in the case of a fungal infection, a fungus involved in the infection can be treated using the antifungal agent in smaller quantities, or can be treated with an antifungal agent which is not therapeutically effective when used in the absence of the efflux pump inhibitor. Thus, this method of treatment is especially appropriate for the treatment of infections involving fungal strains which are difficult to treat using an antifungal agent alone due to a need for high dosage levels (which can cause undesirable side effects), or due to lack of any clinically effective antifungal agents. However, it is also appropriate for treating infections involving microbes which are susceptible to particular antifungal agents as a way to reduce the dosage of those particular agents. This can reduce the risk of side effects, but can also reduce the selection effect for highly resistant microbes resulting from the consistent high level use of a particular antifungal agent. In particular embodiments, various antifungal agents, including the azole class of antifungal agents, can be used. In particular embodiments an antibiotic of the above classes can be, for example, one of the following: terbinafine or an azole antimicrobial agent such as fluconazole, itraconazole, ketoconazole.
In a further related aspect, this invention includes a method for prophylactic treatment of an animal, preferably a mammal, or other organism. In this method, an antimicrobial agent, e.g., an antifungal agent, and an efflux pump inhibitor as described above is administered to an animal or other organism at risk of a microbial infection, e.g., a fungal infection, or for which it is desirable to prevent the establishment of a microbe, e.g., a fungus.
In the context of the response of a microbe (or other cell), such as a fungus, to an antimicrobial agent, the term xe2x80x9csusceptibilityxe2x80x9d refers to the sensitivity of the microbe for the presence of a compound such as an antimicrobial agent. So, stated in the context of microbes, to increase the susceptibility means that the microbe will be inhibited by a lower concentration of the antimicrobial agent in the medium surrounding the microbial cells. This is equivalent to saying that the microbe is more sensitive to the antimicrobial agent. In most cases the minimum inhibitory concentration (MIC) of that antimicrobial agent will have been reduced.
In another related aspect, the invention provides a method for treating a mammal, e.g., a human, suffering from a cancer by administering a milbemycin or milbemycin-type compound or derivative as described above and a second compound as described for inhibiting the growth of a cancer cell above.
As used herein, the term xe2x80x9ctreatingxe2x80x9d refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. The term xe2x80x9cprophylactic treatmentxe2x80x9d refers to treating an organism, such as a human patient, who is not yet infected, but who is susceptible to, or otherwise at risk of, a particular infection. The terms xe2x80x9csusceptiblexe2x80x9d and xe2x80x9cat riskxe2x80x9d do not refer to the status of organisms of that type generally, but rather refers to a significantly enhanced risk. Such risk may, for example, be due to a specific exposure to a particular potentially infective agent, to a generally weakened physical condition, or immune system deficiency. Preferably, for humans, the enhanced risk is sufficient such that a prudent doctor familiar with the treatment of the potential infection would find prophylactic treatment medically warranted. The term xe2x80x9ctherapeutic treatmentxe2x80x9d refers to administering treatment to a patient already bearing or suffering from an infection. Thus, in preferred embodiments, xe2x80x9ctreatingxe2x80x9d is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of an efflux pump inhibitor preferably in combination with a second compound, e.g., an antifungal (or antimicrobial) agent in combination (either simultaneously or serially).
By xe2x80x9ctherapeutically effective amountxe2x80x9d or xe2x80x9cpharmaceutically effective amountxe2x80x9d is meant amounts individually of an efflux pump inhibitor and/or a second compound, such as an antimicrobial agent, as disclosed for this invention, which have a therapeutic effect, which generally refers to the inhibition to some extent of the normal metabolism of partiuclar cells, such as microbial cells causing or contributing to a microbial infection. The doses of efflux pump inhibitor and antimicrobial agent (or similarly efflux pump inhibitor and anticancer agent) which are useful in combination as a treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means those amounts of efflux pump inhibitor and/or antimicrobial agent (or other second compound) which, when used in combination, produce the desired therapeutic effect as judged by clinical trial results and/or model animal infection studies. In particular embodiments, the efflux pump inhibitor and antimicrobial agent (or other second compound) are combined in predetermined proportions and thus a therapeutically effective amount would be an amount of the combination. This amount and the amount of the efflux pump inhibitor and second compound (e.g., antimicrobial agent) individually can be routinely determined by one of skill in the art, and will vary, depending on several factors, such as the particular cells or microbial strain involved and the particular efflux pump inhibitor and second compound (e.g., antimicrobial agent) used. This amount can further depend upon the patient""s height, weight, sex, age and medical history. For prophylactic treatments, a therapeutically effective amount is that amount which would be effective if a microbial infection existed or other condition to be treated existed (e.g., a cancer).
A therapeutic effect relieves, to some extent, one or more of the symptoms of the infection or other condition, and includes curing an infection or other condition. xe2x80x9cCuringxe2x80x9d means that the symptoms of active infection or condition are eliminated, including the elimination of excessive numbers of viable microbes of those involved in the infection or of other cells involved in causing a condition in which the elimination or reduction of certain cells is desirable. However, certain long-term or permanent effects of the infection or condition may exist even after a cure is obtained (such as extensive tissue damage).
The term xe2x80x9cmicrobial infectionxe2x80x9d refers to the presence of a microbe in or on tissues of a host animal, e.g., a mammal. Preferably, but not necessarily, this will be the presence of pathogenic microbes. It can include the growth of microbes which are normally present in or on the body of a mammal, preferably including cases of excessive growth, and other situations where the elimination of the presence of the microbe is desirable (e.g., sub-clinical infections) (or prevention of establishment of the presence of the microbe). Usually, but not necessarily, a microbial infection will be a situation in which the presence of a microbial population(s) is damaging or potentially damaging to a host mammal. Thus, an animal is xe2x80x9csufferingxe2x80x9d from a microbial infection when excessive numbers of a microbial population are present in or on the animal""s body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mammal. As an example, this description specifically applies to a fungal infection.
The term xe2x80x9cbearing a microbial infectionxe2x80x9d indicates that a particular microbial population is present in or on the body of an animal. It is not necessary that the presence of the microbes be damaging to the animal.
The term xe2x80x9cadministrationxe2x80x9d or xe2x80x9cadministeringxe2x80x9d refers to a method of giving a dosage of an antimicrobial pharmaceutical composition to a mammal, where the method is, e.g., topical, oral, intravenous, intraperitoneal, or intramuscular. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the microbe involved, and the severity of an actual microbial infection.
The term xe2x80x9cmammalxe2x80x9d is used in its usual biological sense. Thus, it specifically includes humans, dogs, and cats, but also includes many other species. The term xe2x80x9cwarm-blooded animalxe2x80x9d has its usual biological meaning.
In a related aspect, the invention also provides methods of inhibiting growth of a microbe, e.g., a fungus, by contacting the microbe with an efflux pump inhibitor active on a CDR1, CDR2, BEN, or FLU1 efflux pump or a homolog thereof. Preferably the microbe is also contacted with an antimicrobial, e.g., antifungal agent. In preferred embodiments, the microbe, efflux pump inhibitor, anti microbial agent pharmaceutical compositions, and method of contacting are as described for aspects above or otherwise described herein.
Likewise, in another related aspect, the invention provides a method for prophylactic or therapeutic treatment of a microbial infection of an organism, e.g., a plant or an animal such as a mammal like a human, by administering to the organism a therapeutic amount of an efflux pump inhibitor, where the efflux pump inhibitor is effective to inhibit the activity of at least one of a CDR1, CDR2, BEN, or FLU1 efflux pump or homolog, and preferably of a plurality of such pumps. Preferably, an antimicrobial agent is also administered. Preferred embodiments involve microbes, antimicrobial agents, efflux pump inhibitors, methods of administering the compounds, pharmaceutical compositions, and organisms to be treated as described for aspects above or otherwise described herein.
Stating that the efflux pump inhibitor is xe2x80x9ceffective to inhibit the activityxe2x80x9d of an efflux pump means that the compound at least slows the efflux pump action of the pump in a specific manner, but does not imply that every activity of the pump or of a pump component is necessarily inhibited.
In another related aspect, the invention provides a pharmaceutical composition which includes a milbemycin-type compound and a pharmaceutically acceptable carrier or excipient, where the composition is formulated for administration in conjunction with an antimicrobial agent. Thus, the antimicrobial agent may be separate or may be included in the pharmaceutical composition. In preferred embodiments, the milbemycin-type compound is a compound as indicated in the aspects described above.
Also, in preferred embodiments, the pharmaceutical composition includes an antimicrobial agent, preferably an antifungal agent, preferably an azole antifungal agent, e.g., fluconazole, itraconazole, and ketoconazole, or terbinafine, which is effluxed by an efflux pump which can be inhibited by a milbemycin or milbemycin-type compound in the composition.
In further related aspects, the invention provides a formulation or pharmaceutical composition which includes a milbemycin-type compound and an antimicrobial agent. In preferred embodiments, the milbemycin-type compound is a compound as described above. Also in preferred embodiments, the antimicrobial agent is an antifungal agent, preferably fluconazole, itraconazole, ketoconazole, or terbinafine.
In another aspect, the invention provides a method of screening for efflux pump inhibitors by determining whether the growth of a microbial strain is inhibited to a greater extent in the presence of a combination of a test compound and a sub-inhibitory concentration of an antimicrobial agent than in the presence of the sub-inhibitory concentration of the antimicrobial agent alone. The microbial strain expresses an efflux pump (at least one) which exports the antimicrobial agent, and the efflux pump is a CDR1, CDR2, BEN, or FLU1 efflux pump or a homolog of one of those pumps or a plurality of such pumps. The microbes are cultured under conditions (including a sub-inhibitory concentration of an antimicrobial agent) such that the microbes will grow unless inhibited by the presence of the test compound. Thus, a reduction in growth of the strain in the presence of the test compound as compared to the absence of the test compound is indicative that the test compound is an efflux pump inhibitor.
In preferred embodiments, the microbial strain is a recombinant Saccharomyces cerevisiae strain, for example, a strain expressing a recombinant CDR1, CDR2, BEN, or FLU1 efflux pump from Candida albicans. Also in preferred embodiments, the microbial strain is a fungal strain, such as a Candida species, for example, Candida albicans. In other preferred embodiments, the microbial strain overexpresses one or more of the efflux pumps. In addition, while the screening method can be carried out using a variety of different antimicrobial agents, in preferred embodiments, the antimicrobial agent is fluconazole.
In another aspect, the invention provides a method for preparing a pharmaceutical composition by identifying an efflux pump inhibitor. The identification includes screening for a compound active on a CDR1, CDR2, BEN, or FLU1 efflux pump or a homolog. The screening can, for example, be performed by the above screening method. A compound identified through such screening may be used directly in following steps, or may be used as a hit compound which is used in medicinal chemistry to prepare and identify a compound having more desirable therapeutic properties. A compound so identified is synthesized in an amount sufficient to provide a therapeutic effect when administered to an animal suffering from a microbial infection where the efflux pump inhibitor enhances the susceptibility of the microbial cells to an antimicrobial agent, preferably an antimicrobial agent for which the efflux pump inhibitor has been shown to enhance the susceptibility of cells. The method can also involve combining the efflux pump inhibitor with an antimicrobial agent and/or with a pharmaceutially acceptable carrier or excipient. The identification can, for example, involve microbial cells and/or antimicrobial agents as otherwise described herein. For example, an antimicrobial agent can be terbinafine or an azole antimicrobial agent such as fluconazole, itraconazole, or ketoconazole.
Other features and advantages of the invention will be apparent from the following Detailed Description of the Preferred Embodiments and the accompanying drawing and from the claims.